PROJECT SUMMARY The PD-L1/PD-1 immune checkpoint pathway, consisting of the inhibitory receptor PD-1 on T cells, and its ligand PD-L1 on tumor cells and antigen presenting cells (APCs), is a major mechanism for tumors to escape immune attack. Blocking antibodies of PD-1 and PD-L1 have demonstrated unprecedented clinical activities against an array of human cancers, yet durable benefit is limited to a small subset of patients. Expanding the clinical benefit to a larger population of patients has met critical challenges due to our incomplete understanding of the PD- L1/PD-1 pathway and how their blockade antibodies work. Our long-term goal is to fill these mechanistic gaps through the use of novel, unique and robust approaches. We recently uncovered two novel aspects of PD-L1/PD- 1 signaling. Intracellularly, we showed that the T cell costimulatory receptor CD28 is a primary target of PD-1 associated phosphatases. Extracellularly, we discovered that PD-L1 can be neutralized in cis by PD-1 expressed on the same cells, i.e., APCs. In this proposal, we will integrate and extend these two prior findings to elucidate a poorly defined extracellular crosstalk between PD-L1/PD-1 and CD28 pathways. Our preliminary experiments revealed that the CD28 ligand B7.1 binds and neutralizes PD-L1 in cis, but not in trans. Guided by this new finding, we propose to pursue three specific aims to determine the biochemical and functional consequences of the B7.1/PD-L1 cis-interaction and how this interaction might underlie unreported mechanisms of actions for PD- L1 and PD-1 blockade antibodies. In Aim-1, we will determine whether B7.1 neutralizes the ability of PD-L1 to bind and activate PD-1 through cis-interaction. In Aim-2, we will ask the reciprocal question of whether cis-PD- L1 inhibits the ability of B7.1 to bind and activate CD28. In Aim-3, we will use well-defined co-culture assays and mouse tumor models to determine the effects of PD-1 and PD-L1 blockade antibodies in the context of B7.1/PD- L1 cis-interaction. The key for achieving these goals is to decouple and quantitate cis and trans interaction at the cell-cell interface. To this end, we plan to integrate classical approaches and our recently developed membrane reconstitution, live cell imaging (TIRF) and cell-specific antibody blockade assays. Completion of the project will reveal in-depth insights into the regulatory mechanism of the PD-L1/PD-1 immune checkpoint axis, its crosstalk with the B7/CD28 pathway, and the mechanism of actions of therapeutic antibodies. These mechanistic insights will likely yield novel targets and biomarkers of PD-1 targeted cancer immunotherapy.